Primary cilia control oligodendrocyte precursor cell proliferation in white matter injury via Hedgehog-independent CREB signaling.

Remyelination after white matter injury (WMI) often fails in diseases such as multiple sclerosis because of improper recruitment and repopulation of oligodendrocyte precursor cells (OPCs) in lesions. How OPCs elicit specific intracellular programs in response to a chemically and mechanically diverse environment to properly regenerate myelin remains unclear. OPCs construct primary cilia, specialized signaling compartments that transduce Hedgehog (Hh) and G-protein-coupled receptor (GPCR) signals. We investigated the role of primary cilia in the OPC response to WMI. Removing cilia from OPCs genetically via deletion of Ift88 results in OPCs failing to repopulate WMI lesions because of reduced proliferation. Interestingly, loss of cilia does not affect Hh signaling in OPCs or their responsiveness to Hh signals but instead leads to dysfunctional cyclic AMP (cAMP)-dependent cAMP response element-binding protein (CREB)-mediated transcription. Because inhibition of CREB activity in OPCs reduces proliferation, we propose that a GPCR/cAMP/CREB signaling axis initiated at OPC cilia orchestrates OPC proliferation during development and in response to WMI.

The ancestral ESCRT protein TOM1L2 selects ubiquitinated cargoes for retrieval from cilia.

Many G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as hedgehog morphogens. Lysine 63-linked ubiquitin (UbK63) chains mark GPCRs for regulated removal from cilia, but the molecular basis of UbK63 recognition inside cilia remains elusive. Here, we show that the BBSome-the trafficking complex in charge of retrieving GPCRs from cilia-engages the ancestral endosomal sorting factor target of Myb1-like 2 (TOM1L2) to recognize UbK63 chains within cilia of human and mouse cells. TOM1L2 directly binds to UbK63 chains and the BBSome, and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin, and the GPCRs SSTR3, Smoothened, and GPR161 inside cilia. Furthermore, the single-cell alga Chlamydomonas also requires its TOM1L2 ortholog in order to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.

MRAP2 regulates energy homeostasis by promoting primary cilia localization of MC4R.

The G protein-coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor-associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.

The gymnastics of intraflagellar transport complexes keeps trains running inside cilia.

The assembly and signaling properties of cilia rely on intraflagellar transport (IFT) trains moving proteins into, within, and out of cilia. A flurry of near-atomic models of the multiprotein complexes that make up IFT trains has revealed new conformational changes, which may underlie the switch between anterograde and retrograde intraflagellar transport.

Shedding of ciliary vesicles at a glance.

Cilia sense and transduce sensory stimuli, homeostatic cues and developmental signals by orchestrating signaling reactions. Extracellular vesicles (EVs) that bud from the ciliary membrane have well-studied roles in the disposal of excess ciliary material, most dramatically exemplified by the shedding of micrometer-sized blocks by photoreceptors. Shedding of EVs by cilia also affords cells with a powerful means to shorten cilia. Finally, cilium-derived EVs may enable cell-cell communication in a variety of organisms, ranging from single-cell parasites and algae to nematodes and vertebrates. Mechanistic understanding of EV shedding by cilia is an active area of study, and future progress may open the door to testing the function of ciliary EV shedding in physiological contexts. In this Cell Science at a Glance and the accompanying poster, we discuss the molecular mechanisms that drive the shedding of ciliary material into the extracellular space, the consequences of shedding for the donor cell and the possible roles that ciliary EVs may have in cell non-autonomous contexts.

Time-resolved proteomics profiling of the ciliary Hedgehog response.

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia.

Regulated trafficking of G protein-coupled receptors (GPCRs) controls cilium-based signaling pathways. ß-Arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs, but the functional interplay between ß-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with ubiquitin chains comprising K63 linkages (UbK63) in a ß-arrestin-dependent manner before BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a UbK63-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3, and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.

Near-atomic structures of the BBSome reveal the basis for BBSome activation and binding to GPCR cargoes.

Dynamic trafficking of G protein-coupled receptors (GPCRs) out of cilia is mediated by the BBSome. In concert with its membrane recruitment factor, the small GTPase ARL6/BBS3, the BBSome ferries GPCRs across the transition zone, a diffusion barrier at the base of cilia. Here, we present the near-atomic structures of the BBSome by itself and in complex with ARL6GTP, and we describe the changes in BBSome conformation induced by ARL6GTP binding. Modeling the interactions of the BBSome with membranes and the GPCR Smoothened (SMO) reveals that SMO, and likely also other GPCR cargoes, must release their amphipathic helix 8 from the membrane to be recognized by the BBSome.

The Molecular Architecture of Native BBSome Obtained by an Integrated Structural Approach.

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.

Effects of α-tubulin acetylation on microtubule structure and stability.

Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.

Establishing and regulating the composition of cilia for signal transduction.

The primary cilium is a hair-like surface-exposed organelle of the eukaryotic cell that decodes a variety of signals - such as odorants, light and Hedgehog morphogens - by altering the local concentrations and activities of signalling proteins. Signalling within the cilium is conveyed through a diverse array of second messengers, including conventional signalling molecules (such as cAMP) and some unusual intermediates (such as sterols). Diffusion barriers at the ciliary base establish the unique composition of this signalling compartment, and cilia adapt their proteome to signalling demands through regulated protein trafficking. Much progress has been made on the molecular understanding of regulated ciliary trafficking, which encompasses not only exchanges between the cilium and the rest of the cell but also the shedding of signalling factors into extracellular vesicles.

The molecular machines that traffic signaling receptors into and out of cilia.

Cilia are surface-exposed organelles that dynamically concentrate signaling molecules to organize sensory, developmental and homeostatic pathways. Entry and exit of signaling receptors is germane to the processing of signals and the molecular machines for entry and exit have started to emerge. The IFT-A complex and its membrane recruitment factor Tulp3 complex promotes the entry of signaling receptors into cilia while the BBSome and its membrane recruitment factor Arl6GTP ferry activated signaling receptors out of cilia. Ciliary exit is a surprisingly complex process entailing passage through a first diffusion barrier at the transition zone, diffusion inside an intermediate compartment and crossing of a periciliary diffusion barrier. The two barriers may organize a privileged compartment where activated signaling receptors transiently reside.

BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone.

A diffusion barrier at the transition zone enables the compartmentalization of signaling molecules by cilia. The BBSome and the small guanosine triphosphatase Arl6, which triggers BBSome coat polymerization, are required for the exit of activated signaling receptors from cilia, but how diffusion barriers are crossed when membrane proteins exit cilia remains to be determined. In this study, we found that activation of the ciliary G protein-coupled receptors (GPCRs) Smoothened and SSTR3 drove the Arl6-dependent assembly of large, highly processive, and cargo-laden retrograde BBSome trains. Single-molecule imaging revealed that the assembly of BBSome trains enables the lateral transport of ciliary GPCRs across the transition zone. However, the removal of activated GPCRs from cilia was inefficient because a second periciliary diffusion barrier was infrequently crossed. We conclude that exit from cilia is a two-step process in which BBSome/Arl6 trains first move activated GPCRs through the transition zone before a periciliary barrier can be crossed.

A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies.

Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

BORC Regulates the Axonal Transport of Synaptic Vesicle Precursors by Activating ARL-8.

Axonal transport of synaptic vesicle precursors (SVPs) is essential for synapse development and function. The conserved ARF-like small GTPase ARL-8 is localized to SVPs and directly activates UNC-104/KIF1A, the axonal-transport kinesin for SVPs in C. elegans. It is not clear how ARL-8 is activated in this process. Here we show that part of the BLOC-1-related complex (BORC), previously shown to regulate lysosomal transport, is required to recruit and activate ARL-8 on SVPs. We found mutations in six BORC subunits-blos-1/BLOS1, blos-2/BLOS2, snpn-1/Snapin, sam-4/Myrlysin, blos-7/Lyspersin, and blos-9/MEF2BNB-cause defects in axonal transport of SVPs, leading to ectopic accumulation of synaptic vesicles in the proximal axon. This phenotype is suppressed by constitutively active arl-8 or unc-104 mutants. Furthermore, SAM-4/Myrlysin, a subunit of BORC, promotes the GDP-to-GTP exchange of ARL-8 in vitro and recruits ARL-8 onto SVPs in vivo. Thus, BORC regulates the axonal transport of synaptic materials and synapse formation by controlling the nucleotide state of ARL-8. Interestingly, the other two subunits of BORC essential for lysosomal transport, kxd-1/KXD1 and blos-8/Diaskedin, are not required for the SVP transport, suggesting distinct subunit requirements for lysosomal and SVP trafficking.

Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.

Microtubules acquire resistance from mechanical breakage through intralumenal acetylation.

Eukaryotic cells rely on long-lived microtubules for intracellular transport and as compression-bearing elements. We considered that long-lived microtubules are acetylated inside their lumen and that microtubule acetylation may modify microtubule mechanics. Here, we found that tubulin acetylation is required for the mechanical stabilization of long-lived microtubules in cells. Depletion of the tubulin acetyltransferase TAT1 led to a significant increase in the frequency of microtubule breakage. Nocodazole-resistant microtubules lost upon removal of acetylation were largely restored by either pharmacological or physical removal of compressive forces. In in vitro reconstitution experiments, acetylation was sufficient to protect microtubules from mechanical breakage. Thus, acetylation increases mechanical resilience to ensure the persistence of long-lived microtubules.

Tubulin acetylation protects long-lived microtubules against mechanical ageing.

Long-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on Lys40 of α-tubulin (αK40), acetylation takes place after stabilization and does not protect against depolymerization. Instead, αK40 acetylation has been proposed to mechanically stabilize microtubules. Yet how modification of αK40, a residue exposed to the microtubule lumen and inaccessible to microtubule-associated proteins and motors, could affect microtubule mechanics remains an open question. Here we develop FRET-based assays that report on the lateral interactions between protofilaments and find that αK40 acetylation directly weakens inter-protofilament interactions. Congruently, αK40 acetylation affects two processes largely governed by inter-protofilament interactions, reducing the nucleation frequency and accelerating the shrinkage rate. Most relevant to the biological function of acetylation, microfluidics manipulations demonstrate that αK40 acetylation enhances flexibility and confers resilience against repeated mechanical stresses. Thus, unlike deacetylated microtubules that accumulate damage when subjected to repeated stresses, long-lived microtubules are protected from mechanical ageing through their acquisition of αK40 acetylation. In contrast to other tubulin post-translational modifications that act through microtubule-associated proteins, motors and severing enzymes, intraluminal acetylation directly tunes the compliance and resilience of microtubules.

Fifteen years of research on oral-facial-digital syndromes: from 1 to 16 causal genes.

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.